-1-

Structure, Volume 20

Supplemental Information

Interaction of the Mediator Head Module

with RNA Polymerase II

Gang Cai, Yuriy L. Chaban, Tsuyoshi Imasaki, Julio A. Kovacs, Guillermo Calero,

Pawel A. Penczek, Yuichiro Takagi, and Francisco J. Asturias

Inventory of Supplemental information

Supplemental Methods, Monte Carlo Docking Analysis

Figure S1, related to Figure 1C

Figure S2, related to Figure 2C, 2D

Figure S3, related to Figure 2C, 2D

Figure S4, related to Figure 3A

Figure S5, related to Figure 3A

Figure S6, related to Figure 3A

Figure S7, related to Figure 3A

Figure S8, related to Figure 4

Figure S9, related to Figure 4

Figure S10, related to Figure 5A

Figure S11, related to Figure 6A, 6B

Cai, et al.

-1-

SUPPLEMENTAL METHODS

Monte Carlo Docking Analysis

We developed a novel Monte Carlo Docking approach that provides an estimate of

the precision for docking of X-ray segments into a cryo-EM map. The method is based

on the realization that the resolution estimate routinely used in the single particle EM

field, the Fourier Shell Correlation (FSC), also provides an estimate of the Spectral-

Signal-to-Noise Ratio (SSNR) of an EM map, by virtue of the relation:

SSNR s FSC s 1 FSC s , (Eq.S1)

where s s is the modulus of the spatial frequency. Therefore, if the original, noise-

free map F were known, one could use a simple additive linear image formation model:

G s F s N s , (Eq.S2)

where N is noise, and use the definition of SSNR:

SSNR s F s 2

N s 2, (Eq.S3)

to generate maps that have the same amount of noise, thus the same SSNR and

resolution, as the observed map by:

G s F s M s F s 2

SSNR s , (Eq.S4)

where M is a is a random realization of white noise, with M 1. Note that involves

additional rotational averaging that results in a 1D profile of the respective entity. Since

the original map F is not known, in the proposed procedure we replace it by the

observed map G (this is justified by the fact that the amount of noise in 3D EM maps is

relatively small).

The described relations allow us to generate multiple version of the reconstructed

3D map of a complex, which have different noise components but have the same SSNR

Cai, et al.

-2-

as the original experimental map. Thus, computationally, we create a situation

equivalent to performing multiple experimental determinations of the structure of the

complex under study. The availability of multiple versions of the EM map allows us to

dock a given X-ray segment into all of them, and compute the statistics of the resulting

set of orientation parameters. Ultimately, the procedure yields standard deviation values

for the shift and rotation-induced errors associated with docking of an X-ray segment.

Finally, we generate "clouds", i.e., 3D objects that outline regions of space within which

a docked segment can be found with a predetermined probability. These clouds reflect

the statistical significance of the fit and provide an intuitive, visual assessment of

docking errors. 

Cai, et al.

-3-

SUPPLEMENTAL FIGURES

FIGURE S1, related to Figure 1C. Analysis of transcripts from the synthetic DNA promoter in the Head-mPIC complex. (A) Length (L) and intensity normalization factor (Norm; dependent on the number of incorporated radioactive UTPs) for each transcript band. (B) Histogram showing the absolute numbers of transcript/template (x103) corresponding to different bands.

FIGURE S2, related to Figure 2C, 2D. Initial clustering of cryo-EM images. Initial class averages obtained through reference-free alignment and clustering of images obtained from Head-mPIC cryo-EM samples. Many of the averages resemble projections of the RNAPII structure, whereas others (highlighted by the red square outlines) clearly display non-RNAPII density.

FIGURE S3, related to Figure 2C, 2D. Multi-reference classification and reference-free alignment of cryo-EM images. Competitive classification was used to separate cryo-EM images into groups according to orientation, and the absence (A), or presence (B) of the Head module. Shown here are class averages obtained after reference-free alignment within each group obtained through multi-reference classification. These reference-free class averages (rows marked by arrowheads) match closely corresponding projections of refined 3D structures of Headless and Head-complexes.

FIGURE S4, related to Figure 3A. Cryo-EM maps derived from Headless and Head-containing particle images in the Head-mPIC data set. (A) The structure calculated from Headless particles (purple mesh) closely resembles a molecular model of RNAPII (solid orange) generated from its X-ray structure. (B) The structure generated from particle images in which the Head was present is reminiscent of the polymerase structure but also contains considerable additional density.

FIGURE S5, related to Figure 3A. Refinement of a Head-mPIC cryo-EM map starting from a very low resolution reference. (A) Different views of a ~50 Å reference used to refine a cryo-EM map of the Head-mPIC complex (B; same orientations as the reference) using SPARX from a subset (~27K images) of the Head-containing images in the Head-mPIC data set. The SPARX-refined cryo-EM map closely resembles the SPIDER-refined map (Figure S4B) obtained after refinement of all (~51K) Head-containing images in the Head-mPIC data set.

 

FIGURE S6, related to Figure 3A. SPARX clustering analysis of the Head-mPIC images. Head-containing images in the Head-mPIC data set were aligned and clustered using a new reference-free, iterative algorithm. The resulting, independently-derived

Cai, et al.

-4-

reference-free class averages (left images in each column) correspond well to re-projections of the Head-mPIC 3D cryo-EM map (right images in each column).

FIGURE S7, related to Figure 3A. EM analysis of the Head-mPIC complex. (A) Angular distribution of refined orientations for Head-containing particle images. (B) Fourier Shell Correlation analysis indicates a resolution (at 0.5 cut-off level) of ~16 Å for the Head-mPIC complex cryo-EM map.

FIGURE S8, related to Figure 4. Monte Carlo docking analysis of the Head-mPIC cryo-EM map. (A) Mean positions and 1 clouds for each RNAPII module derived from docking of the modules into noise-corrupted realizations of the cryo-EM map. On the left column, mean positions (colored surfaces) are shown within the cryo-EM map of the Head-mPIC complex (shown as a semi-transparent gray mesh surface). The X-ray model of RNAPII (teal ribbons) is also included for reference. On the right column, the same mean module positions are shown inside their corresponding 1 clouds (shown as mesh surfaces, Rpb4-Rpb7 were left out to facilitate display of the clamp position). (B) Positions and 1σ clouds for the shelf and Rpb8 modules. The 1σ clouds are displaced with respect to the initial positions of the modules, indicating the likelihood of significant (> 10 À) displacement, and possibly explaining poor definition of the corresponding portion of the cryo-EM map. (C) Comparison of the RNAPII X-ray structure with the RNAPII conformation derived from docking analysis. If the core module (the largest and most stable portion of the RNAPII structure) in the RNAPII X-ray model (teal ribbons) is matched to the corresponding portion of the cryo-EM map, the X-ray positions of the core, jaw, shelf, and Rpb8 modules lie within the respective 1σ clouds. However, the X-ray positions of the clamp and Rpb4-Rpb7 lie well outside the respective 1σ clouds, indicative of significant large changes in the position of the clamp and Rpb4-Rpb7 upon interaction of RNAPII with the Head module.

FIGURE S9, related to Figure 4. Changes in the RNAPII structure. (A) Approximate movement undergone by individual RNAPII structural modules upon interaction with the Head module is illustrated by comparing their relative positions (in gray) in the RNAPII X-ray structure with their approximate positions after docking into the cryo-EM map of the Head-mPIC complex. Views of each module correspond to the Front view of the RNAPII structure, except where indicated in parenthesis (R=Right view, T=Top view). Movement of the modules (indicated by the curved arrows) is analogous to that observed upon comparison of different RNAPII crystal forms, but much larger, particularly for the clamp and Rpb4-Rpb7. (B) Changes in the position of the RNAPII clamp observed by comparison of different crystal structures, and after interaction with the Head module. Matching positions of the shelf module in X-ray structures of RNAPII alone (PDB accession code 1i3q) and interacting with an open promoter complex (PDB accession code 3gtg) illustrates that the clamp can swing out by as much as ~25 (left panel). In contrast, comparing the position of the fully closed clamp with that estimated for the clamp in the Head-mPIC cryo-EM map shows a change of ~45 (right panel). This large change in clamp position results from extensive interaction of the clamp with the Head and to Head-induced changes in the position of the Rpb4-Rpb7 polymerase subunit complex that normally limits clamp mobility.

Cai, et al.

-5-

FIGURE S10, related to Figure 5. Codimensional Principal Component Analysis (CD-PCA) of the Head-mPIC complex data. A stratified image resampling approach was used to generate 12,000 resampled Head-mPIC volumes that guided statistical analysis of the images in the Head-mPIC dataset. (A) Five Head-mPIC maps generated through CD-PCA (B) The validity of the original CD-PCA maps is evidenced by the observation that their features are preserved in maps obtained after images within each CD-PCA group were re-aligned. (C) Using the five original CD-PCA maps as initial references for multi-reference refinement results in five new image groups and five new refined maps that still preserve the characteristics of the original CD-PCA maps. Moreover, nearly half of the images in the new groups remain associated to the group to which they were first assigned by CD-PCA. Densities marked with (I) are apparent in the final Head-mPIC cryo-EM map. The density marked (II) is also present in that map but is only observed at thresholds slightly lower than the one used for rendering the volume.

FIGURE S11, related to Figure 6A, 6B. Distribution of non-RNAPII density in the Head-mPIC EM structure and comparison with the position of TFIIF-interacting areas in the RNAPII structure. (A) Areas on the surface of a molecular map of RNAPII (derived from the X-ray structure of the enzyme) located within 20 Å of residues reported to interact with TFIIF are colored as indicated. (B) Non-RNAPII densities in the EM structure of the Head-mPIC complex can be tentatively identified as specific TFIIF domains based on their proximity to segments of the RNAPII structure involved in interaction with TFIIF domains. The Tfg1-Tfg2 dimerization domain (DD), Tfg2 wing-helix (Tfg2 WH), and Tfg2 C-terminus (Tfg2 C-ter), can be tentatively identified in the Head-mPIC cryo-EM map.

Cai, et al.

-6-

FIGURE S1

 

Cai, et al.

-7-

Figure S2

 

Cai, et al.

-8-

Figure S3

 

Cai, et al.

-9-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure S4

 

Cai, et al.

-10-

Figure S5

 

Cai, et al.

-11-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure S6

Cai, et al.

-12-

Figure S7

 

Cai, et al.

-13-

Figure S8

 

Cai, et al.

-14-

Figure S9

Cai, et al.

-15-

Figure S10

 

Cai, et al.

-16-

Figure S11